New polychlorinated bibenzyls from Rhododendron minutiflorum

Five new polychlorinated bibenzyls (1–5) along with 3 known compounds (6–8) were isolated from the stems and leaves of Rhododendron minutiflorum. The chemical structures of all the isolates were determined by spectroscopic methods, and compounds 1 and 2 were further verified by single-crystal X-ray diffraction analyses. Compounds 1–5 were halogenated compounds which bear three to five chlorine atoms in their chemical structures. Biologically, compounds 2, 5 and 6 showed varying degrees of toxicity toward the Asian citrus psyllid (Diaphorina citri) with LD50 values 27.15, 17.02 and 16.20 mg/L, respectively. These values were comparable to the positive control matrine (LD50 = 11.86 mg/L), which were calculated using observations on day 6. Meanwhile, compound 4 had α-glucosidase inhibitory activity with IC50 value of 17.87 ± 0.74 μM. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s13659-022-00364-x.

Compound 4 was obtained as a white amorphous powder and had a molecular formula of C 14  Moreover, the key HMBC correlations (Fig. 2) supported that the positions of C-3, C-4, C-3′, C-4′ and C-5′ in 4 were substituted by two hydroxyl groups and three chlorine atoms. The aforementioned analyses suggested that the structure of 4 closely resembled 2, except for the absence of a chlorine atom at C-2 in 4. Thus, 4 was characterized as a polychlorinated bibenzyl and assigned as 4,3′,5′-trichloro-3,4′-dihydroxybibenzyl.
Compound 5, a white amorphous powder, had a molecular formula of C 15 (Tables 1 and  2) and 2D NMR data ( Fig. 2) showed that the structure of 5 was similar to 4, differing only by the hydroxy at C-3 in 4 being replaced by a methoxy in 5, which was confirmed by the HMBC correlation ( Fig. 2) from δ H 3.86 (-OMe) to δ C 155.0 (C-3). Thus, the structure of 5 was elucidated as shown in Fig. 1 and named as 4,3′,5′-trichloro-4′-hydroxyl-3-methoxybibenzyl.

Insecticidal activity and α-glucosidase inhibitory activity
In order to evaluate the insecticidal activities of all the isolated compounds, their toxicities on D. citri Kuwayama were tested. With compound 4 as the only exception, compounds 1-3 and 5-8 exhibited different toxicities to D. citri compared to the 10% methanol or DMSO controls. Compounds 2, 5 and 6 showed remarkable insecticidal activities, with LD 50 values of 27.15, 17.02 and 16.20 mg/L, respectively. While the LD 50 of matrine was 11.86 mg/L (Table 3). Interestingly, on the 4th day after the test started, the corrected mortality rate of citrus psyllid is higher than 50% when the compound concentration is higher than 50 mg/L, indicating that compounds 2, 5 and 6 had insecticidal activity comparable to positive control matrine (Fig. 4). These data could provide reference and scientific basis for the further development and utilization of new pesticides against the Asian citrus psyllid D. citri. Besides, the α-glucosidase inhibitory activity of compounds 1-8 were detected by PNPG method, with acarbose served as a positive control ( Table 4). The results showed that compounds 1, 3-8 gave different degrees of inhibitory activity with IC 50 values from 17.87 to 82.84 μM (the IC 50 of acarbose was 3.07 × 10 -3 μM). Furthermore, the preliminary structure-activity relationships (SAR) analyses showed that compounds with two chlorine atoms at C-2 and C-4 or C-4 and C-6 in ring A possessed insecticidal activity while compounds with three chlorine atoms at C-2, C-4 and C-6 in ring A had no more insecticidal activity, such as the insecticidal activity

General experimental procedures
The instruments, materials and reagents used in this study were consistent with those reported in the literature [13,20].

Plant materials
Dr.

Single-crystal X-ray diffraction data of 1
Colorless crystals of 1: Moiety formula were C 14

Single-crystal X-ray diffraction data of 2
Colorless crystals of 2: Molecular formula was C 14  Compounds 1 and 2 are colorless crystals that were obtained by vapor diffusion in MeOH solvent at room temperature. The intensity data were acquired in the way described in the previous literatures [21][22][23]. These data have been deposited in the Cambridge Crystallographic Data Centre as supplementary publications numbers CCDC 2105148 for 1, 2105091 for 2. Free copies of the data can be obtained from CCDC through www. ccdc. cam. ac. uk.

The biological assay
In November 2021, D. citri were captured from 5-yearold citrus trees (Citrus reticulata Blanco CV' Shatangju) in an orchard in Lingchuan, Guilin, China (25° 41 N, 110° 36 E). To avoid accidental injury, all the D. citri were carefully collected and cultured. The insecticidal activities of eight compounds were evaluated using methods described in the literature [24,25] with slightly modified. In brief, D. citri were treated with compounds 1-8 and the positive control at concentrations of 100, 50, 25, 12.5 and 6.25 mg/L or 200, 100, 50, 25 and 12.5 mg/L. The blank solution was prepared with 10% methanol or DMSO in distilled water, respectively. Finally, mortality was calculated every 24 h starting on the second day of the trial. The toxicity data were statistically analyzed by Probit analyses, and the value of 50% lethal dose (LD 50 ) were calculated.
According to the previously reported method [13], all the isolated compounds were screened for α-glucosidase inhibitory activity. Acarbose (Med Chem Express, NJ, USA, HY-B0089) was used as a positive control.

Conclusion
Five new polychlorinated bibenzyls (1)(2)(3)(4)(5) and three known bibenzyls (6)(7)(8) were isolated from the stems and leaves of R. minutiflorum. All the chemical structures were determined by spectroscopic methods, and the structures of compounds 1 and 2 were further verified by the single-crystal X-ray diffraction analyses. Compounds 1-5 were rare halogenated compounds which bear three to five chlorine atoms. As we know, it's the first time to find polychlorinated bibenzyls from Rhododendron plants. The insecticidal activity against the Asian citrus psyllid D. citri and the inhibitory activity on α-glucosidase of all the isolated compounds were evaluated. It is concluded that compounds 2, 5 and 6 from R. minutiflorum Hu exhibited potential insecticidal activity against the Asian citrus psyllid D. citri, and compounds 4 showed potent inhibitory activity against α-glucosidase.